Freitas Cleverson D T, Souza Diego P, Grangeiro Thalles B, Sousa Jeanlex S, Lima Isis V M, Souza Pedro Filho N, Lima Cristiano S, Gomes Alexandre D'Emery S, Monteiro-Moreira Ana C O, Aguiar Tawanny K B, Ramos Márcio V
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Campus do Pici, Bloco 907, Fortaleza, Ceará CEP 60451-970, Brazil.
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Campus do Pici, Bloco 907, Fortaleza, Ceará CEP 60451-970, Brazil.
Int J Biol Macromol. 2023 Dec 1;252:126529. doi: 10.1016/j.ijbiomac.2023.126529. Epub 2023 Aug 24.
Although latex fluids are found in >20,000 plant species, the biochemical composition and biological function of their proteins are still poorly explored. Thus, this work aimed to conduct a proteomic analysis of Cryptostegia grandiflora latex (CgLP) for subsequent purification and characterization of an antifungal protein. After 2D-SDS-PAGE and mass spectrometry, 27 proteins were identified in CgLP, including a polygalacturonase inhibitor, cysteine peptidases, pathogenesis-related proteins (PR-4), and osmotins. Then, two osmotin isoforms (CgOsm) were purified, and a unique N-terminal sequence was determined (ATFDIRSNCPYTVWAAAVPGGGRRLDRGQTWTINVAPGTA). The PCR products revealed a cDNA sequence of 609 nucleotides for CgOsm, which encoded a polypeptide with 203 amino acid residues. The structure of CgOsm has features of typical osmotin or thaumatin-like proteins (TLPs), such as 16 conserved Cys residues, REDDD and FF motifs, an acidic cleft, and three main domains. Atomic force microscopy (AFM) and bioinformatics suggested that CgOsm is associated with three chain units. This result was interesting since the literature describes osmotins and TLPs as monomers. AFM also showed that Fusarium falciforme spores treated with CgOsm were drastically damaged. Therefore, it is speculated that CgOsm forms pores in the membrane of these cells, causing the leakage of cytoplasmic content.
尽管在20000多种植物物种中都发现了乳胶液，但对其蛋白质的生化组成和生物学功能仍知之甚少。因此，本研究旨在对大花羊角拗乳胶（CgLP）进行蛋白质组学分析，以便随后纯化和鉴定一种抗真菌蛋白。经过二维SDS-PAGE和质谱分析，在CgLP中鉴定出了27种蛋白质，包括一种多聚半乳糖醛酸酶抑制剂、半胱氨酸蛋白酶、病程相关蛋白（PR-4）和渗透素。然后，纯化了两种渗透素同工型（CgOsm），并确定了一个独特的N端序列（ATFDIRSNCPYTVWAAAVPGGGRRLDRGQTWTINVAPGTA）。PCR产物显示CgOsm的cDNA序列为609个核苷酸，编码一个含有203个氨基酸残基的多肽。CgOsm的结构具有典型渗透素或类甜蛋白（TLP）的特征，如16个保守的半胱氨酸残基、REDDD和FF基序、一个酸性裂隙以及三个主要结构域。原子力显微镜（AFM）和生物信息学表明CgOsm与三个链单元相关。这一结果很有趣，因为文献中描述渗透素和TLP为单体。AFM还显示，用CgOsm处理的镰孢镰刀菌孢子受到了严重损伤。因此，推测CgOsm在这些细胞的膜上形成孔，导致细胞质内容物泄漏。
